While abnormalities in monoamine metabolism have been investigated heavily per potential roles in the mechanisms of depression, the contribution of amino acid metabolism in the brain remains not well understood. In additional, roles of the hypothalamus-pituitary-adrenal axis in stress-regulation mechanisms have been of much focus, while the contribution of central amino acid metabolism to these mechanisms has not been well appreciated. Therefore, whether depression-like states affect amino acid metabolism and their potential roles on stress-regulatory mechanisms were investigated by comparing Wistar Kyoto rats, which display depression-like behaviors and stress vulnerability, to control Wistar rats. Brain amino acid metabolism in Wistar Kyoto rats was greatly different from normal Wistar rats, with special reference to lower cystathionine and serine levels. In addition, Wistar Kyoto rats demonstrated abnormality in dopamine metabolism compared with Wistar rats. In the case of stress response, amino acid levels having a sedative and/or hypnotic effect were constant in the brain of Wistar Kyoto rats, though these amino acid levels were reduced in Wistar rats under a stressful condition. These results suggest that the abnormal amino acid metabolism may induce depression-like behaviors and stress vulnerability in Wistar Kyoto rats. Therefore, we hypothesized that abnormalities in amino acid and monoamine metabolism may induce depression, and amino acid metabolism in the brain may be related to stress vulnerability.

I.c.v. injection of L-ornithine has been shown to have sedative and hypnotic effects on neonatal chicks exposed to acute stressful conditions. To clarify the mechanism, we conducted three experiments under strengthened stressful conditions with corticotropin-releasing factor (CRF). In Experiment 1, the effect of i.c.v. injection of CRF, L-ornithine (0.5 μmol) or CRF with L-ornithine on the stressful response of chicks was investigated. Compared with the vehicle control, CRF increased distress vocalizations and the time spent in active wakefulness. L-ornithine increased the time spent in sleeping posture, even following stimulation with CRF. In Experiment 2, dose-dependent effects of L-ornithine were investigated using i.c.v. administration with vehicle, CRF alone or CRF plus L-ornithine (0.125, 0.25 or 0.5 μmol). L-ornithine decreased the CRF-stimulated distress vocalizations in a dose-dependent manner. In Experiment 3, the chicks were injected i.c.v. with either CRF, CRF plus L-ornithine (0.5 μmol), CRF plus the γ-aminobutyric acid (GABA)A receptor antagonist picrotoxin or L-ornithine with picrotoxin. The sedative and hypnotic effects induced by L-ornithine were blocked with co-administration of picrotoxin. These results suggest that L-ornithine could attenuate CRF-stimulated stress behaviors acting at GABAA receptors.

Precision oncology with next generation sequencing (NGS) using tumor tissue with or without blood has begun in Japan. Tumor molecular profiling tests are available, including the OncoGuide™ NCC Oncopanel System and FoundationOne ® CDx (F1CDx). Our purpose was to identify potentially actionable genetic alterations in breast cancer with this comprehensive tumor profiling test. We enrolled 115 patients with pathologically diagnosed advanced or metastatic breast cancer. Comprehensive tumor genomic profiling, microsatellite instability, and tumor mutational burden (TMB) were determined using F1CDx. Testing was successful in 109/115 cases (94.8%). Clinically actionable alterations were identified in 76% of advanced breast cancer patients. The most frequent short variants were in TP53 (48.6%), PIK3CA (38.5%), GATA3 (11.0%), PTEN (11.0%), and BRCA1 (10.1%), and structural variants were in ERBB2 (24.8%), MYC (21.1%), RAD21 (21.1%), CCND1 (11.9%), FGF19 (10.1%), and PTEN (10.1%). Regarding human epidermal growth factor receptor (HER)2 status, 106/109 samples (97.2%) were concordant between F1CDx and HER2 testing with immunohistochemistry/fluorescence in situ hybridization. However, ERBB2 amplification was newly detected in four samples and ERBB2 mutations were detected in five HER2‐negative breast cancer samples. Oncogenic BRCA mutations were found in three samples with F1CDx among 27 germline testing‐negative samples. The mean TMB in all samples was 6.28 mut/Mb and tended to be higher in luminal B and triple‐negative breast cancer (mean = 8.1 and 5.9 mut/Mb, respectively) compared with other subtypes. In conclusion, we established a system for precision oncology and obtained preliminary data with NGS as the first step. The information in this clinical sequencing panel will help guide the development of new treatments for breast cancer patients.

Intracerebroventricular injection of L-ornithine has demonstrated sedative and hypnotic effects in neonatal chicks exposed to acute stressful conditions. However, whether orally administered L-ornithine can reduce acute mental stress remains to be defined. To clarify the nutritional importance of L-ornithine in controlling the stress response, in Experiment 1 we first investigated whether orally administered L-ornithine can be transported into the brain of mice. Mice were orally administered L-ornithine (3 mmol/water 10 ml/kg, per os). L-Ornithine levels were significantly elevated in the cerebral cortex and hippocampus at 30 and 60 minutes post-administration. In Experiment 2, the effect of orally administered L-ornithine (0, 0.1875, 0.75 and 3 mmol/water 10 ml/kg, per os) on anxiety-like behavior in mice exposed to the elevated plus-maze test was examined at 30 minutes post-administration. There was a significant increase in the percentage of time spent and entries in the open arms in the group receiving 0.75 mmol of L-ornithine compared to the control group. Furthermore, locomotion activity in a novel environment was not significantly changed between the control group and 0.75 mmol of L-ornithine group in Experiment 3. Therefore, it appears that orally administrated L-ornithine is bioavailable to the rodent brain and reduces anxiety-like behavior as demonstrated by the elevated plus-maze test.

Purpose T-box transcription factor 21 (T-bet), which is the master regulator of effector T-cell activation, is derived by stimulation of T-cell receptors. In this study, we focused on T-bet and examined the function of activated T cells. Methods This study included 242 patients with primary triple-negative breast cancer (TNBC) who underwent resection without neoadjuvant chemotherapy between January 2004 and December 2014. The immunohistochemistry scoring for CD8 and T-bet expression on tumor-infiltrating lymphocytes (TILs) was defined as ≥ 30 per 6.25 × 10 −3 mm 2 . Results Of the 242 TNBC cases, CD8 was positively expressed in 127 (52.5%) tumors, and T-bet was positively expressed in 67 (27.7%) tumors. T-bet expression was significantly correlated with CD8 expression ( p < 0.0001). Patients with T-bet + tumors had longer overall survival (OS) compared with patients with T-bet − tumors ( p = 0.047). The combination of CD8 + and T-bet + was associated with a better recurrence-free survival (RFS) and OS compared to CD8 + /T-bet − tumors ( p = 0.037 and p = 0.024, respectively). Adjuvant chemotherapy provided significantly greater benefit to patients with T-bet + tumors ( p = 0.031 for RFS, p = 0.0003 for OS). Multivariate analysis revealed that T-bet expression on TILs was an independent and positive prognostic indicator (HR = 0.36, 95% confidence interval (CI) 0.12–0.94, p = 0.037 for RFS, HR = 0.30, 95% CI 0.07–0.95, p = 0.039 for OS). Conclusions OS was significantly improved for patients with high T-bet-expressing TILs in TNBC. Thus, T-bet may be a predictive indicator for survival and various immunotherapy strategies in TNBC. Electronic supplementary material The online version of this article (10.1007/s10549-019-05256-2) contains supplementary material, which is available to authorized users.

IntroductionThe optimal suction pressure during endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) remains to be determined. The aim of this study was to compare suction pressures for performance in collecting sufficient tissue specimens from mediastinal and hilar lymph nodes during EBUS-TBNA.MethodsRetrospective analysis of consecutive patients with mediastinal and hilar lymphadenopathy who underwent EBUS-TBNA over a 3-year period. Results from patients who underwent EBUS-TBNA using a dedicated 20-mL VacLoc (Merit Medical Systems, Inc, South Jordan, UT) syringe (conventional method, group C) were compared with results from patients in whom a disposable 30-mL syringe (high pressure group, group H) was used. The yield for sufficient histologic specimen retrieval and amount of tissue obtained were compared between the 2 groups.ResultsOf 178 patients who underwent EBUS-TBNA, 131 had lung cancer confirmed by EBUS-TBNA: 35 in group C and 96 in group H. There were 7 patients in group C and 6 in group H who received final diagnoses by cytology alone. There were 28 in group C and 90 in group H who were diagnosed by both cytology and histology. There was a statistically significant difference between the groups in terms of the rate of sufficient sampling for histological specimens (p = 0.04). The H group revealed a tissue area approximately twice that of the C group (p = 0.003). There were no major procedure-related complications in either group.ConclusionHigher suction pressures with larger syringe volumes during EBUS-TBNA may be useful for safely collecting sufficient tissue specimens.

Background It is important to identify biomarkers for triple-negative breast cancers (TNBCs). Recently, pembrolizumab, an immune checkpoint inhibitor (ICI) for programmed cell death 1 (PD-1), was approved as a treatment strategy for unresectable or metastatic tumor with high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency, such as malignant melanoma, non-small cell lung cancer, renal cell cancer and urothelial cancer. In addition, results from clinical trials suggested that ICI was a promising treatment for TNBCs with accumulated mutations. However, the frequency of MSI in Japanese TNBCs still remains unclear. We aimed to analyze the presence of MSI-H in TNBCs as a biomarker for ICI therapy. Methods In this study, we retrospectively evaluated the MSI of 228 TNBCs using an innovative method, MSI Analysis System Version 1.2 (Promega), consisting of 5 microsatellite markers: BAT-26, NR-21, BAT-25, MONO-27 and NR-24 without a normal tissue control. Results Among 228 tumors, 222 (97.4%) were microsatellite stable, 4 (1.7%) low-frequency MSI and 2 (0.9%) MSI-H, respectively. Two MSI-H tumors were potentially aggressive pathologically as indicated by nuclear grade 3 and high Ki-67 (> 30%), and were classified as basal-like and non-BRCA-like, but were not consistent regarding tumor-infiltrating lymphocytes, CD8 and PD-L1 expression. Conclusions Although we found that MSI-H was uncommon (0.9%) in TNBCs, potential targets for ICIs exist in TNBCs. Therefore, MSI-H breast cancer patients should be picked up using not only conventional methods but also platforms for comprehensive genomic profiling.

In a previous study, we confirmed that orally administered L-ornithine can be transported into the brain of mice. In addition, orally administered L-ornithine, within a limited dose range, had an anxiolytic-like effect in the elevated plus-maze test. However, the mechanism by which orally administered L-ornithine reduced the stress response in mice is still unclear. Experiment 1 determined whether orally administered L-ornithine could reduce the stress-induced activation of hypothalamic-pituitary-adrenal axis. Mice were orally administered L-ornithine (0, 0.75, 1.5 and 3 mmol/10 ml/kg, p.o.), and restrained for 30 min from 30 min post administration. There was a significant decrease in the corticosterone levels in the group receiving 0.75 mmol of L-ornithine compared to the control group. In Experiment 2, the effect of orally administered L-ornithine (0 and 0.75 mmol/10 ml/kg, p.o.) on endogenous monoamine release was investigated using in vivo microdialysis. Only the monoamines metabolites 5-hydroxyindoleacetic acid (5-HIAA), dihydroxyphenylacetic acid (DOPAC) and homovallinic acids (HVA) were detected in the present study. Dialysate concentrations of 5-HIAA, DOPAC and HVA were not significantly changed immediately after administration of L-ornithine and restraint stress. In conclusion, changes of corticosterone concentrations by orally administered L-ornithine were not related to alterations in brain monoamine metabolisms.